PROJECT SUMMARY The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, is estimated to inhabit the mouths of approximately 30% of the human population. It is associated with a variety of periodontal diseases, but A. actinomycetemcomitans is most predominantly associated with localized aggressive periodontitis (LAP), where it is detected in about 85% of the LAP cases. To evade the immune response of the host, the bacterium secretes the leukotoxin, LtxA. LtxA is a member of the repeat-in-toxin (RTX) family of toxins and exhibits a specificity for human WBCs driven by its recognition of the lymphocyte function-associated antigen 1 (LFA-1) integrin and its affinity for cholesterol. Both components are required for LtxA cytotoxicity, therefore, it is hypothesized that LFA- 1 and cholesterol play a combined role for LtxA activity. In our preliminary data, we have successfully purified LFA-1 from cell membranes and verified its functionality with regard to LtxA binding. We plan to reconstitute this transmembrane protein into proteoliposomes as part of a multifaceted approach using natural and model biological systems to determine how individual interactions of LtxA with LFA-1 and cholesterol lead to cell death. In Aim 1, we will investigate how LtxA is moved into the lipid raft compartment on the cell membrane by identifying changes in requirements for LtxA-LFA-1 and LtxA-cholesterol binding inside and outside of lipid raft domains. After LtxA has moved into the lipid rafts, it is endocytosed where it subsequently moves into the lysosome. In Aim 2, we will identify changes to LtxA and LFA-1 binding facilitated by the acidic environment inside the lysosomal lumen. At the conclusion of this project, we will have extensively expanded our knowledge on the roles of LFA-1 and cholesterol in the cytotoxic activity of LtxA, which will be useful for the future development of new therapeutic strategies against A. actinomycetemcomitans infections. We also expect these results to have broader implications through applying similar approaches to identify specific mechanisms of cytotoxic activity of other RTX toxins.